The long-range goal of the proposed research is to understand the roles of the extracellular matrix (ECM) proteins fibronectin (FN) and laminin (LAM) in peripheral nervous system (PNS) development. ECM molecules regulate cell shape, differentiation, and movement during embryogenesis. In the PNS, ECM molecules appear to be involved in neural crest migration and development and regeneration of neuromuscular junctions. Growth cones of nerve fibers migrating from the neural tube or peripheral ganglia traverse substrate pathways that are also likely to contain adhesive molecules. The adhesive glycoproteins FN and LAM are good candidates for mediating particular stages of PNS cell migration and differentiation, due to their locations in embryos and interactions with PNS cells in vitro. First, what functional regions of FN and LAM mediate neuron-substratum adhesion and neurite extension? Two regions of FN have these activities and have revealed neuronal type-specific interactions with the FN molecule. Functional regions of LAM are less well-defined, but may also promote cell type-specific behavior. Proteolytic fragments of FN and LAM will be used, in conjuction with antibodies, to evaluate 1) the functional distinctions between the regions of FN that interact with cells, 2) whether cell surface heparan sulfate mediates certain of these interactions, and 3) how each domain contributes to mediation of cell behavior by the intact FN molecule. In addition, the role of growth cone adhesion in neurite extension on FN fragments will be studied. Second, how are FN and LAM (and specific regions of each) used by cells in the developing PNS? Approaches will be 1) immunocytochemistry to reveal temporal and spatial associations between cells and ECM molecules, 2) immunolabeling for TEM and SEM to identify the cells producing LAM and FM and their proximity to growth cones, and 3) inhibition, with antibodies, of neurite extension on naturally-occurring matrices and in vivo to probe the relative roles of each molecule. Third, how do defined regions of FN and LAM contribute to migration and differentiation of neural crest cells? Do crest cell subpopulations attach to, and/or move upon, certain regions of these molecules at particular stages of differentiation? Conversely, do defined regions of ECM molecules exert differentiative effects on crest cell subpopulations? (To be studied in collaboration with Dr. James Weston's lab) With our background in cell-ECM interactions and the tools now available to us, we will be able to evaluate precisely the roles of FN and LAM in PNS development.